9 July 2011

Chemical tagging for live cell fluorescence imaging

Fluorescent proteins are not the only way to examine live cells by fluorescence imaging methods. Fluorescent chemical tagging is becoming a more and more attractive alternative accompanied by much less perturbation of cell behavior.

Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins (FPs) are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. Compared to FPs, chemical tags are smaller and so, the perturbation of the natural behavior of a protein of interest is less. Chemical tags ideally can be combined with arbitrary biophysical probes.

In a review article, Richard Wombacher, Heidelberg University, Germany, and Virginia W. Cornish, Columbia University, New York, USA, report on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. They discuss selected experiments in which chemical tags have been successfully applied to live cell imaging.

Three general strategies have been developed for protein labeling in living cells, all based on the specific interaction between a genetically encoded tag and a small molecule. In the case of fluorescent labeling, the small molecule is either fluorescent itself or covalently linked to a fluorophore. The binding of a fluorescent label to the tag sequence occurs either by formation of a covalent bond (by self-modification), a non-covalent high affinity binding, or the formation of a covalent bond mediated by an enzyme (enzyme mediated tagging). The polypeptide tag can be an intact protein or a short peptide, expressed as fusion with the protein of interest (POI). The Cornish group, for example, invented the first noncovalent protein tag which exploits the high affinity binding of folate analogs to the protein dihydrofolate reductase from Escherichia coli.

In recent years, some of the chemical tagging methods have surpassed the proof-of-principle stage and have been used to study the localization and dynamics of proteins in living cells, especially in experiments that cannot be easily performed with fluorescent proteins. In chromophore assisted light inactivation, for example, chemical tags offer the ideal technology to localize photosensitizers in close proximity to the target protein. When directly attached to the POI, the damage from the generated reactive oxygen species to other proteins will most likely be minimized.

For Ca2+-imaging experiments, chemical methods have been developed to combine the advantages of genetically encoded tags and chemical Ca2+-indicators. Chemical tags are ideally suited to localize chemical sensors to a specific organelle or protein. For example, it had been possible to measure calcium concentrations close to ion channels in cells.

Recently, it could be shown that chemical tagging strategies can be ideal platforms to introduce fluorophores that have the photophysical characteristics to allow for super-resolution (SR) microscopy with live cells. For example, a noncovalent tag has been developed that is suitable for SR-imaging based on direct stochastic optical reconstruction microscopy (dSTORM). Wombacher et al. successfully labeled a histone protein. Applying dSTORM imaging allowed the identification of histones within the nucleoprotein complex. Furthermore, they could exploit the label to follow nucleosome movements in living cells.

R. Wombacher et al., J. Biophotonics 4(6), 391–402 (2011), DOI 10.1002/jbio.201100018

Round Robin Experiment

Raman spectroscopy has already proved its effectiveness in many cases for medical diagnostics such as for cancer, cardiovascular diseases and infections. However, there are no standards in the different working groups, e.g. for sample preparation, implementation of the Raman experiments, spectra pre-treatment, data evaluation, etc.In a round robin experiment, the required groundwork will take place in order to define standardised Raman measurement methods, which will be fundamental for establishing Raman spectroscopy for clinical diagnostic procedures.

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